Despite decades of research, the morbidity and mortality of necrotizing enterocolitis (NEC) remain unchanged. An altered intestinal microbiome and intestinal inflammation may predispose prematures to NEC. Probiotics may protect the intestines from NEC, however, they have to be delivered in high numbers with repeated dosing for any effect. We have developed a novel, tunable delivery system in which Lactobacillus reuteri (Lr) administered in a protective biofilm on biocompatable microspheres provides persistent probiotic benefits from just a single dose, greatly reducing experimental NEC. Our long-term goal is to identify novel strategies to protect neonates from NEC. The current overall objective is to identify a novel probiotic-based therapy protective against experimental NEC. Our central hypothesis is that administering Lr in a biofilm state with biocompatible microspheres that can be loaded with beneficial luminal cargo significantly improves its ability to colonize the intestines, reduce intestinal inflammation, and decrease the incidence of NEC. The rationale is that identification of an optimal probiotic delivery system will lead to maximally beneficial treatment of NEC. Elucidating the attributes of Lr that prevent NEC will also provide insight into factors essential for NEC development. We will objectively test our central hypothesis by pursuing the following specific aims: 1) To determine the impact of our Lr formulation in protection of the intestines from NEC. 2) Tuning the beneficial activities (biofilm formation, antimicrobial and anti-inflammatory effects) of Lr to optimize our therapeutic formulation. 3) To determine whether tuning the properties of Lr alters its ability to protect the intestines from NEC. Expected outcomes include identification of the most effective Lr delivery system to reduce NEC, and determination of the relative importance of anti-inflammatory and anti-microbial effects of Lr in protection from NEC. The significance is a better understanding of the effects of probiotics on protection of intestines, allowing the best design of clinical probiotic-based therapies for NEC. This will have a positive impact in terms of providing improved therapeutic interventions for patients at risk of developing NEC, in addition to fundamentally advancing our understanding of the mechanisms by which Lr exerts its beneficial intestinal effects. This research is innovative because it involves a novel probiotic formulation that: (i) requires only a single dose for beneficial effects on NEC and (ii) is a platform by which mechanisms of probiosis can be studied through targeted delivery of prebiotic substrates in well defined microspheres.